Structural and regulatory studies on protein components of the E. coli sugar transport system known as the phosphoenolpyruvate:sugar phosphotransferase system (PTS) continued. New studies, in collaboration with the Clore laboratory (NIDDK), have elucidated the three-dimensional structure, by NMR, of the 48-kDa IIAMannose-HPr complex. IIAMannose is dimeric and has two symmetrically related binding sites per dimer for HPr. A convex surface on HPr interacts with a deep groove at the interface of the two subunits of IIAMannose. The interaction surface on IIAMannose is predominantly helical. The binding sites on the two proteins are complementary in terms of shape and distribution of hydrophobic, hydrophilic and charged residues. The active site histidines of IIAMannose and HPr are in close proximity. We have also been carrying out structural and functional studies on a pathway, paralagous to the sugar transport system, known as the nitrogen regulatory PTS. The protein currently under study is the paralog of the sugar transporter IIAGlucose, referred to as IIANitrogen. In collaboration with the Wang laboratory (Omaha, Nebraska), we have elucidated, by NMR, the solution structure of IIANitrogen as well as its interaction with its partner protein NPr, a paralog of HPr of the sugar transport system. The diffusion coefficient indicates that the functional form of IIANitrogen is a monomer (~18 kDa) in solution. Thus, the dimeric structure of the protein found in the crystal is an artifact of crystal packing. The residual dipolar coupling data are consistent with the structure in solution matching that of molecule A of the crystal structure. Chemical shift mapping identified the surface on IIANitrogen for NPr binding. In collaboration with the Seok laboratory (Seoul, Korea), we have investigated the biological function of IIANitrogen. We carried out phenotype microarray analysis on a mutant deleted for the gene expressing the first enzyme of the nitrogen regulatory PTS (Enzyme INitrogen). The findings of these studies, suggesting resistance to the growth inhibitory effects of certain peptides, led to growth studies with a mutant deleted for the gene encoding IIANitrogen. These studies revealed that the IIANitrogen mutant was extremely sensistive to leucine-containing peptides. The toxicity of leucine-containing peptides was found to be due to leucine and the dephospho-form of IIANitrogen was found to be necessary to neutralize leucine toxicity. Further studies showed that the dephospho-form of IIANitrogen is required for derepression of the ilvBN operon encoding acetohydroxyacid synthetase, which catalyzes the first step common to the biosynthesis of the branched-chain amino acids.